Insertion sequences (IS) are examples of a large group of translocatable DNA elements which have the ability to move from one chromosomal site to another independent of normal recombinational mechanisms. They play an important role in bacterial evolution and may be representative of a more universal class of elements involved in site-specific gene rearrangements and amplification, and perhaps control of gene expression in both prokaryotes and eukaryotes. I propose to use two approaches to elucidate the mechanism of the site-specific and rec-independent insertion and excision events associated with the element, IS1. a) The first involves characterization of the DNA regions which participate in translocation, namely the ends of IS1, the chromosomal sites of IS1 insertion, these same sites after IS1 excision, and the end points of IS1-induced deletions. Five independent IS1 insertions in the gal operon of E. coli will be analyzed. A method has been devised for mapping with restriction endonucleases and identifying the DNA regions of interest. Specific DNA fragments will be isolated and sequenced using the direct chemical modification methods of Maxam and Gilbert. Comparison of the DNA sequences obtained should indicate the recognition sites of enzymes involved in IS1 recombinational phenomena. b) It seems likely that translocation of IS1 requires the production of an IS1-encoded protein. The second approach, therefore, involves the examination of DNA fragments lacking any natural bacterial promoters for specific IS1-promoted transcription in vitro. A further aspect of the expression of IS1 is the strong polar effect that it exerts on genes downstream from the insertion site, independent of the orientation of the inserted DNA. By isolation mutants of IS1 which have lost the polarity effect, I hope to gain some understanding of IS1-induced transcriptional termination.